Method for biochemical analysis of dna and arrangement associated therewith

ABSTRACT

A system with immobilized DNA is used in the fields of medicine, environmentology or criminology as an analytical tool in the analysis of nucleic acid. The immobilized DNA is provided with a biocatalytically active marker, such as an enzyme, an with an inhibitor substance which reversibly inhibits catalytic activity, or in addition to the immobilized biocatalytic marker, the immobilized DNA is provided with a substance which can reversibly inhibit the catalytic activity of the marker. Alternatively, an immobilized biocatalytically active marker can be provided with DNA as a scavenger which includes a substance as an inhibitor which can reversibly inhibit the activity of the marker. In another alternative, it is possible to use a complex including a molecule binding double-stranded DNA and a substance as an inhibitor which can reversibly inhibit the activity of the marker by interacting with the immobilized biocatalytically active marker. In all cases, the inhibitor or compound including an inhibitor and a molecule which can bind double-stranded DNA interacts with the biocatalytically active marker and defines the inactive state of the system. When the DNA, which is to be analyzed, is bonded, especially hybridized, to the DNA scavengers, the interaction between the biocatalytically active marker and the inhibitor is cancelled as a result of the formation of the double strand. The system is thus shifted from a first state into a second state defining the active state. A carrier with integrated microelectrodes is provided in the associated device, whereby the enzyme is either immobilized therein or is contained in a polymer network in the vicinity of the microelectrodes.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/DE03/01479 which has an Internationalfiling date of May 8, 2003, which designated the United States ofAmerica and which claims priority on German Patent Application number DE102 20 935.9 filed May 10, 2002, the entire contents of which are herebyincorporated herein by reference.

FIELD OF THE INVENTION

The invention generally relates to a method for biochemically analyzingDNA. In addition, the invention generally relates to an associatedarrangement for implementing this method.

The fields of application of the invention are, in particular, medicine,environmental analysis and forensics. In this connection, an enzyme, asan immobilized biocatalytically active label, an immobilized DNA and animmobilized substance (inhibitor), which is able to inhibit the activityof the enzyme reversibly, are used as tools for analyzing nucleic acids.

In the present context, DNA (deoxyribonucleic acid) is understood asmeaning a deoxyribonucleic acid and its structural analogs. These are,in particular, PNA (peptide nucleic acid), “caged” DNA, RNA (ribonucleicacid) and all 2′-substituted DNA derivatives.

The invention is not applicable to ribozymes. In this connection, thereader is referred to the publication “Catalytic Molecular Beacons” inCHEMBIOCHEM 2001, 2, 411-415.

The aim of the present developments is that of performing a molecularanalysis at the level of DNA and/or gene expression, in the latter caseby way of analyzing cDNA (complementary DNA) in particular. This makesit possible to identify and type hereditary material-containingpathogens, such as bacteria and viruses or the like, and to clarify anyresistances which may be present. Furthermore, it makes it possible todetect organisms in environmental analysis, foodstuffs technology andagriculture. In addition to this, the use of this type of DNA analysisin medicine offers the possibilities of rapidly performing hereditarydisease, predisposition and/or tumor diagnosis as well as monitoringtherapy.

BACKGROUND OF THE INVENTION

In accordance with the prior art, immobilized DNA is used as ananalytical tool in analyzing the sequences of nucleic acids. For this,synthetic DNA having a length of up to 100 nucleotide building blocks(DNA oligonucleotides) is covalently linked to a suitable surface by wayof an active group. The surfaces which are used can be silicates, metallayers, for example gold or the like, or else a variety of polymerlayers.

The latter technology is highly developed and makes it possible tospecifically immobilize DNA oligo-nucleotides of defined sequence onareas having a diameter of up to a few μm or in volumes having a contentof up to a few nl. Complementary DNA molecules from the sample can thenbe bound to these areas or volumes which are occupied by what are termedthe DNA catchers. The specificity of this binding is defined by the ruleof DNA complementary base pairing. When a range of DNA molecules havingdifferent sequences is present in the analyte solution, those DNAmolecules which conform best with the base pairing rules, and whichrelease the greatest quantity of energy in connection with the complexformation, will bind to the catcher.

Specific selection, what is termed stringency, of the externalconditions, such as temperature, ionic strength, etc., during thebinding by means of hybridization results in only the most stablepairings of catcher and analyte DNA, that is those pairings whichconform completely to the base pairing rules, being selectivelyretained.

The latter is the basis of DNA analysis on what are termed DNA chips.The general advantages of such a DNA analysis on chips are to be seen inthe high degree of miniaturization, the synchronization and the highspeed of the overall process as compared with conventional methods.Because of the lower requirement for reagents and sample material, thisis accompanied by a reduction in costs. In addition to this, the use ofDNA chips leads to an increase in the efficiency and precision of theDNA analysis process.

The various types of DNA chip differ, in particular, in the choice ofthe substrate, such as plastic, glass, silicon, etc., in the method ofimmobilization, e.g. gold-thiol coupling, immobilization in gel or thelike, in the technology of the application to the solid surface, such ason-line synthesis, dispensing or the like, and in the nature of thedetection, in particular optical and/or electrochemical, of the DNAinteractions.

The spectroscopic systems in which the DNA to be analyzed is provided,by means of PCR (polymerase chain reaction) or SDA (strand displacementamplification), with a fluorescent reporter group, as explaineddiagrammatically by means of FIG. 1, are the most widespread. In detail,the circles in the figure represent the spectroscopic reporter groupswhich are coupled to the analyte DNA either directly, by way of PCR/SDA,or indirectly, by way of what are termed fluorescent signaloligonucleotides which have been introduced in what is termed a sandwichhybridization assay. After the analyte DNA which does not bind, or onlybinds weakly, has been removed by applying what are termed stringentconditions, e.g. high temperature, low ionic strength, organic solvent,the sites at which the interaction has taken place can be visualized bymeans of fluorescence microscopy, junction-type detectors or a CCDcamera. The regions on the surface at which the interaction betweencatcher and analyte DNA has taken place appear as spots which possessaltered optical properties. Since the positions of the different catcherDNAs on the chip are known, the corresponding complementary DNA which ispresent in the analyte samples can be identified unambiguously.

DNA chips containing some thousand different oligonucleotides/cm² arecommercially available, as are systems for optical analysis. Inparticular, EP 0 745 690 A2 describes optical systems containing probesin which what are termed stem loop structures are refolded byhybridization, with this being detected optically.

In the case of optical detection systems, comparatively complicatedreading and analytical instruments are required, with these instrumentsimmediately primarily restricting the use of the DNA chip technology tospecialized laboratories. It is doubtful whether the DNA analysis ofthis type can be applied broadly in field analysis, e.g. in agriculturalbusinesses, in the foodstuffs industry, in environmental analysis or inproduction-accompanying analysis, or in the case of doctors having theirown independent practices. Simply preparing the samples using PCR or SDAand/or introducing the spectroscopic reporter groups into the analyteDNAs is time-consuming and expensive and may possibly be subject totechnological problems.

Electrochemical methods which detect DNA-DNA interactions offer theadvantage of small, robust, hand-held instruments which are suitable forwhat may possibly be on-site battery operation. Electrochemicaldetermination of the DNA hybridization has thus far in the main made useof the increase in the conductivity of the double-stranded DNA after thehybridization.

Analytical methods which are based on using the conductivity of the DNAfollowing hybridization are not well advanced technically. In thesemethods, powerful electric fields result in DNA damage and consequentlysignal loss.

In addition, the conductivity of the DNA becomes greatly reduced as thelength of the double helix increases. None of the previously employedmethods enables the DNA hybridization to be determined quantitatively.

What are termed redox (re)cycling tests offer a robust approach forsolving the problem of making DNA hybridization accessible to anelectrochemical measurement. In this approach, the hybridization eventbetween bound catcher DNA and biotin-labeled analyte DNA is, forexample, labeled by way of a biotin-streptavidin interaction using anenzyme. The activity of the biocatalyst, e.g. alkaline phosphatase, thenforms a redox-active product, e.g. p-aminophenol, which can betransformed amperometrically at suitable electrodes, e.g. goldelectrodes. As a result of the choice of the special electrode geometry,in particular interdigital electrodes, and of the small electrodedistances of <1 μm, for example, a redox cycling process can start aftersuitable potentials have been applied, with the current of this processbeing a measure of the DNA hybridization event.

A redox cycling system is described, by way of example, in WO 01/75149A2. In this connection, use is made, in particular, of a three-electrodesystem having, for example, interdigital measuring electrodes.

SUMMARY OF THE INVENTION

An object of an embodiment of the invention is to specify an improvedmethod for a DNA label-free biochemical analysis of the DNA-DNAinteraction and to create the associated arrangements.

According to an embodiment of the invention, an object is achieved by asequence of procedural steps. In particular, an implementation of anembodiment of the invention is termed an enzyme switch. An associatedarrangement is further specified in another embodiment.

In the method according to an embodiment of the invention, it isadvantageously possible to provide immobilized DNA, as catcher, with abiocatalytically active label and a substance, as inhibitor, which isable, by interaction with the label, to inhibit its catalytic activityreversibly. Alternatively, it is possible to immobilize a DNA, ascatcher, in the vicinity of the immobilized biocatalytic label, withthis DNA being provided with a substance, as inhibitor, which is able,by interaction with the biocatalytically active label, to inhibit itscatalytic activity reversibly.

Alternatively, an immobilized biocatalytically active label can beprovided with a DNA, as catcher, with this DNA as catcher, with thisDNA, for its part, carrying a substance, as inhibitor, which is able, byinteraction with the label, to inhibit its activity reversibly. In otheralternatives, it is possible to use a complex composed of adouble-stranded DNA-binding molecule and a substance, as inhibitor,which is able, by interaction with the immobilized biocatalyticallyactive label, to inhibit its activity reversibly. When analyte DNA andimmobilized catcher DNA hybridize, this complex binds to the resultingdouble strand and is consequently no longer available for inhibiting thebiocatalytically active label.

In all the alternatives cited, the structure of the catcher DNA, i.e.its partial single-/double-strandedness, enables, in a first inactivestate of the system, the inhibitor and the biocatalyst to interact. Whenthe DNA to be analyzed binds to the catcher DNA because of thecomplementarity, the formation of this double strand abolishes theinteraction between the biocatalyst and the inhibitor or results in theinhibitor being bound to the double strand which has formed. In thisway, the system is switched from the first, inactive state into asecond, active state.

An embodiment of the invention reduces or even eliminates disadvantagesof the prior art. An embodiment of the invention provides, inparticular, for the use of a switchable biocatalyst, namely the enzyme,with the activity of the biocatalyst being controlled and, inparticular, switched by way of the hybridization of the sample DNA tothe catcher DNA.

An arrangement for implementing the method according to an embodiment ofthe invention comprises a support, on which an enzyme is immobilized ata site, a catcher DNA which is immobilized at the site, an inhibitorwhich is covalently linked to the catcher DNA, and a substrate, with, ina first state, the catcher DNA being folded, by way of intramolecularhydrogen bonds, such that the inhibitor inhibits the activity of theenzyme and the substrate is not transformed, and with, in a secondstate, the catcher DNA hybridizing with a DNA to be detected and therebybeing folded such that the inhibitor is separated from the enzyme andthe substrate is transformed.

In an embodiment of the invention, the nucleic acids can be analyzedoptically or electrochemically by way of a hybridization switch. Inparticular, the electrochemical measurement can take placeamperometrically, potentiometrically or conductometrically. This therebymay result in the following substantial advantages as compared with theprior art:

-   -   a label-free read-out method for analyzing DNA is created. Thus,        there is no need, for detecting the hybridization between        catcher DNA and analyte DNA, to introduce any reporter group        into the analyte DNA directly or indirectly by way of a further        hybridization step with a signal oligonucleotide as signal DNA.        This has the advantage that, when the concentration of analyte        DNA is adequate, it is possible to dispense with a        time-consuming and expensive PCR/SDA for introducing a label as        reporter. It is also possible to do without a further        hybridization, which is otherwise necessary in some cases, with        a signal DNA for detecting the catcher/analyte DNA hybridization        as a sandwich assay, thereby markedly simplifying the complexity        of the biochemical detection system and thereby reducing sources        of error.    -   The correlation between the quantity of enzyme product formed        and the quantity of the double-stranded catcher/analyte DNA        makes it possible to evaluate the analyte DNA concentration in        the sample quantitatively.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantages and details of the invention ensue from the followingdescription of illustrated exemplary embodiments, making use of thedrawing in combination with the patent claims. In each case as adiagram,

FIG. 1 shows an analytical system in accordance with the prior art,

FIGS. 2/3, 4/5, 6/7 and 8/9 in each case show systems which involvecontrolling the enzyme activity by means of DNA hybridization and inwhich a double helix is formed,

FIG. 10 shows a plan view of a transducer array together with anenlarged detail for clarifying the construction and production of thecomplete system,

FIG. 11 shows a scheme illustrating the course of a measurement, and

FIG. 12 shows an electrochemical system for analyzing switch functionsin accordance with FIG. 2/3, 4/5, 6/7 or 8/9.

In the figures, the same elements have the same reference numbers. Thefigures are described below, in some cases jointly.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

Reference has already been made to FIG. 1 in the introduction whilediscussing the prior art. In the case of a DNA chip which operates inaccordance with the optical principle, the circles represent fluorescentreporter groups which are coupled to the analyte DNA/signal DNA. Theinformation of interest is obtained by optical interrogation.

The subsequent description of the figures relates initially to FIGS. 2to 9. The same phenomenological principles apply to all these figures.

A support 1 is in each case present as substrate in FIGS. 2 to 9. If anelectrochemical read-out method, in particular redox cycling, is used,the support 1 is a chip having integrated circuits which are not shownhere in detail. These circuits can be analog or digital in design.

FIGS. 2/3, 4/5, 6/7 and 8/9 in each case show control of thebiocatalytic activity by means of DNA hybridization, with thisconsequently effecting a switch. DNA 10 or 10′, with 10 being what istermed a catcher DNA and 10′ being the DNA to be analyzed, abiocatalytically active label 20 and an inhibitor 30, whose interactionis explained below with the aid of alternative examples, are in eachcase present.

The biocatalytically active label 20 is, in particular, an enzyme.However, it can also be a ribozyme.

In FIGS. 2, 4 and 6, the enzyme 20 is inactive. The structure of thecatcher 10, i.e. the partial intramolecular DNA double strand broughtabout by hydrogen bonds 40, enables the inhibitor 30 to interactwith/reversibly bind to the enzyme 20, as the biocatalytically activelabel, and inhibit its activity. The switch is in the inactive state.

The hybridization of the catcher DNA 10 with the analyte DNA 10′ forms aDNA double strand which is composed of catcher DNA 10 and analyte DNA10′. This takes place because the formation of this double strand isenergetically more favorable, due to the higher number of base pairings,i.e. the hydrogen bonds 40, which are formed, than the formation of thepartial intramolecular catcher DNA double strand, which only contains afew hydrogen bonds. The formation of this double strand brings about aconformational change in the catcher which is so powerful that theinteraction of enzyme 20 and inhibitor 30 is weakened such that theinhibitor 30 comes away from the enzyme 20, with the active center ofthe enzyme 20 then being free and the enzyme 20 being active.

The enzyme substrate 50 which is present in the vicinity can now fillthe active center of the enzyme 20. The enzyme 20 is transformed and anoptically or, in particular, electrochemically, i.e. amperometrically,potentiometrically or conductometrically, detectable product will arise.The enzyme 20 is “switched-on”.

The enzyme is consequently active in FIGS. 3, 5 and 7. The switch is inthe active state.

The alternatives depicted in FIGS. 2/3, 4/5, 6/7 and 8/9 relate todifferent variants of the binding/immobilization of biocatalyticallyactive label and/or catcher DNA 10 and of the inhibitor 30.

In FIG. 2 and FIG. 3, both the biocatalytically active label 20 and theinhibitor 30 are bound to the catcher DNA 10, which is fixed to a site 2on the support or chip 1.

As shown in FIG. 4 and FIG. 5, the biocatalytically active label 20 is,in an alternative to FIG. 2/3, immobilized at a site 3 on the chip 1 andboth the catcher DNA 10 and the inhibitor 30 are coupled to it.

As shown in FIG. 6 and FIG. 7, the catcher DNA 10 is, in anotheralternative, bound to a first site 2 on the support or chip 1 while thebiocatalytically active label 20 is bound to a second site 3 on thesupport or chip 1.

As shown in FIG. 8 and FIG. 9, the catcher DNA 10 is, in anotheralternative, fixed, in the inactive state, at the site 2 while thebiocatalytic label is fixed at the site 3. In FIG. 8, the catcher DNA 10is free and single-stranded because the sequence of the catcher is suchthat no intramolecular hydrogen bonds 40 can be formed. In contrast withthe previously described alternatives, what is termed an intercalator60, i.e. a double-stranded DNA-binding molecule, is bonded to theinhibitor 30.

In FIG. 9, an analyte DNA 10′ binds to the catcher DNA 10 with theformation of the hydrogen bonds 40. As a result of the formation of theDNA double strand, the compound or the complex composed of intercalator60 and inhibitor 30 now binds to the double strand. The enzyme 20 isconsequently freely available to the substrate 50 and is active.

In the example shown in FIG. 8/9, the enzyme 20 can also be immobilizedon the support 1 and the catcher DNA 10 can be bound to it. The catcherDNA 10 can just as well be immobilized on the support 1 and the enzyme20 can be bound to it. In this regard, the examples shown in thealternative FIGS. 6/7 and 2/3 take precedence.

In general, immobilization/integration into a polymeric gel matrix canin each case also be used as the binding of the catcher DNA 10 and/or ofthe bio-catalytic label 20 to the chip 1 as support. The gel matrix canbe a hydrogel, which is described elsewhere.

The covalent immobilization of the biocatalytically active label, i.e.at the enzyme, is in all cases effected specifically by way of asuitable amino acid side chain. Advantageously, the enzyme possesses thefollowing properties:

-   -   Either the product or the substrate of the enzymic reaction must        be optically or amperometrically detectable. The phosphatases,        esterases and proteases which catalyze the formation of        phenolates and compounds of the quinone type are particularly        suitable.    -   The enzyme should be composed of a polypeptide chain in order to        ensure the immobilization of the polypeptide chains without any        loss of activity.    -   The enzyme should be sufficiently thermostable to enable DNA-DNA        hybridization to take place over wide temperature ranges.        Enzymes from thermophilic organisms usually satisfy this        condition. Thermo-stable enzymes can have a low specific        activity at room temperatures. This problem can be solved by        means of directed mutagenesis.    -   In order to enable the enzyme activity to be selectively        immobilized and controlled over wide temperature ranges, an        expression system for expressing the enzyme from a recombinant        plasmid should be present.

The production of a transducer system which is designed as a m×n arrayhaving m columns and n lines is described with the aid of FIG. 10:circular analytical positions 101, 101′, etc., which are separated bybarriers 150, are present on a transducer surface 100 which is suitablefor the redox (re)cycling method. Structures having interdigitalelectrodes 110 and, respectively, 120 are located on positions 101,101′, etc., which typically have a diameter of approx. 150 μm and adistance from each other (what is termed pitch) of approx. 200 μm. Theinterdigital electrodes 110 and, respectively, 120 have, in a knownmanner, a comb-like design with electrode fingers 111 and, respectively,121 which have a line and spacing width of not more than 1 μm and whichare advantageously composed of gold. Read-out contacts 160 are arrangedlaterally at the transducer surface 100.

A hydrogel which is not depicted in detail and in which the catcher DNAis anchored covalently by way of a 3′ amino modification is applied tothe analytical positions 101, 101′, etc. At its 5′ end, the catcher DNAcarries an SH group to which the inhibitor of the reporter enzyme, e.g.carboxyl esterase, is bound covalently. An alkyl trifluoromethyl ketone,preferably a trifluoromethyl methyl ketone, is used as the reversibleinhibitor of the esterase.

Catcher DNA and inhibitor are coupled in a suitable manner in accordancewith the following reaction:Oligonucleotide-5′-linker-SH+Br—CH₂—COCF₃→oligonucleotide-5′-linker-S—CH₂—COCF₃

In addition to the complex composed of catcher DNA and inhibitor, thereporter enzyme, preferably a thermostable enzyme which consists of apolypeptide chain, is anchored at each analytical position.Advantageously, the carboxyl esterase from the thermoacidophiliceubacterium Bacillus acidocaldarius (Manco, G., Adinolfi, E., Pisani, F.M., Ottolina, G. Carrera, G. and Rossi, M. 1998, Biochem. J. 332,203-212) is chosen for this purpose. The fact that the X-ray structureof the enzyme is known (De Simone, G., Galdiero, S., Manco G., Lang, D.,Rossi, M., and Pedone, C. 2000, J. Mol. Biol. 303, 761-771) is utilizedfor covalently binding-on the enzyme. This knowledge makes it possibleto use directed mutagenesis to replace a suitable amino acid on thesurface of the enzyme with cysteine or an amino acid, e.g. lysine, whichhas an aminofunctional radical. The enzyme is then bound directly to thegold surface of the interdigital electrodes by way of the SH group ofthe cysteine or else to the particular hydrogel matrix by way of the NH₂group of the aminofunctional radical.

The following applies for operating the switch in accordance with thesequence scheme which is shown in FIG. 11 and which has the constituentsteps a), b), c) and d): the figure shows two adjacent analyticalpositions which are provided with different catcher DNAs. In the groundstate of the system, the catcher DNA at each respective analyticalposition is present, after filling with a suitable buffer solution, in aconformation where the inhibitor is able to bind to the active center ofthe enzyme. The enzyme is inactive; the system is correspondingly in aninactive state as shown in FIG. 11 a).

After DNA to be analyzed has been added and stringent washing has takenplace, a conformational change in the catcher which is so powerful thatthe interaction of the enzyme and inhibitor is weakened such that theinhibitor comes away from the enzyme is only brought about at theanalytical position(s), specifically the left-hand of the two analyticalpositions in FIG. 11, where a stable nucleic acid double strand isformed as a result of the complementarity between the catcher DNA andthe analyte DNA species. The active center of the enzyme is then freeand the enzyme is active; the system is correspondingly in an activestate as shown in FIG. 11 b).

After a suitable enzyme substrate, advantageously the p-aminophenoloctanoyl ester in accordance with step (c), has been added, thesubstrate can fill the active center of the enzyme at the analyticalposition(s), specifically the left-hand position in FIG. 11, at which,as can be seen from constituent FIG. 11 b), a hybridization of analyteDNA and catcher DNA has taken place in accordance with step (d). It isonly at these positions, specifically the left-hand position in FIG. 11,that the substrate is transformed and the amperometrically detectableproduct p-aminophenol can be produced.

The esterase activity is as shown in the following reaction:

In order to amplify the signal, an oxidative or reductive potential isapplied to the different “fingers” 111 and, respectively, 121 of theinter-digital electrodes 110 and, respectively, 120 of a singleanalytical position 101 as shown in FIG. 10. Due to the spacing and linewidths, a redox cycling process then starts at the individual analyticalpositions at which p-aminophenol octanoyl ester has been/is beingconverted to p-aminophenol by means of enzymic activity. The redoxcycling process is to be understood as meaning the oxidation ofp-aminophenol to quinoneimine at the positively polarized electrode andthe reduction of quinoneimine to p-aminophenol at the negativelypolarized electrode. The total current of these redox reactions is afunction of the quantity of hybridized analyte DNA.

FIG. 12 clarifies the detection principle using the redox cyclingprocess, as explained above, and the principle of electrochemicalevaluation. In detail, a redox cycling process is depicted at thesurface of a single analytical position 101 on the chip 1, whichposition is separated off by walls 15, with, in addition to the symbolswhich have already been explained, reference number 80 denoting thequinoneimine and reference number 90 denoting the p-amino-phenol inaccordance with the above structural formula. Microelectrodes 5, 5′ arearranged on the chip 1 with a gm-spacing. The microelectrodes 5, 5′ formpart of the interdigital electrodes 110 and, respectively, 120 havingthe finger electrodes 111 and, respectively, 121 in FIG. 10 and aresupplied with different potentials. Redox currents up into the sub-nanoampere range can be measured at the microelectrodes 5, 5′ by way ofmeasurement electronics using current meters 8 and, respectively, 8′.

A time-dependent measurement signal I=g(t), whose slope S=f(DNA) dependson the DNA to be analyzed, is obtained. This thereby creates a procedurewhich can be used to evaluate the DNA electrochemically. The essentialadvantage of this procedure is that it makes it possible to use DNAsamples which have not previously been modified with a label.

The adjacent measurement positions in FIG. 12 correspond to the singleanalytical positions 101, 101′, etc. as shown in FIG. 10. As describedin detail in connection with the latter figure, they typically have a200 μm grid size, which means that a large number of parallelmeasurements can be carried out on one chip 1.

The above-described examples can be used to produce microchips for ahand-held instrument which is simple to operate and which can be usedfor the defined applications. The replaceable chips have a definedlifetime and can be programmed with different catchers. Since this DNAchip type is a disposable product, a requirement for very high numbersof different DNA chips can be expected. No comparable, simple-to-useinstruments of this type exist on the market.

Exemplary embodiments being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the present invention, andall such modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

1. A method for detecting DNA with use being made of a system employingimmobilized DNAs as an analytical tool, comprising: fixing the enzyme ina stationary manner in the system which is used as an analytical tooland which contains a catcher DNA, an inhibitor and an enzyme, asbiocatalytic label; using the catcher DNA to permit, in a first inactivestate of the system, the inhibitor and enzyme to interact; forming, whenthe DNA to be analyzed is bound to the catcher DNA, a double strand dueto the complementarity and at least one of abolishing and preventing theinteraction between enzyme and inhibitor, wherein in this way, thesystem is switched from the first inactive state to a second, activestate; and measuring the signals of at least one of the active andinactive state via an electrochemically detectable substance whoseconcentration changes due to the enzyme activity.
 2. The method asclaimed in claim 1, wherein the structure of the catcher DNA enables theinhibitor and biocatalyst to interact.
 3. The method as claimed in claim2, wherein immobilized DNA is used as catcher DNA and wherein theimmobilized DNA is provided with the biocatalytically active label and,as inhibitor, a substance which is able, by interaction with the label,to inhibit its catalytic activity reversibly.
 4. The method as claimedin claim 2, wherein an immobilized biocatalytic label is used andwherein a DNA, which is provided with a substance, as inhibitor, whichis able, by interaction with the label, to inhibit its catalyticactivity reversibly, is immobilized, as catcher, in the vicinity of theimmobilized biocatalytic label.
 5. The method as claimed in claim 2,wherein an immobilized biocatalytic label is used and wherein theimmobilized biocatalytically active label is provided with a DNA, ascatcher, which DNA, for its part, carries a substance, as inhibitor,which is able, by interaction with the label, to inhibit its activityreversibly.
 6. The method as claimed in claim 1, wherein use is made ofa complex composed of a double-stranded DNA-binding molecule and aninhibitor substance which is able, by interaction with the immobilizedbiocatalytically active label, to inhibit its activity reversibly, withthe complex being bound, when the immobilized catcher DNA and theanalyte DNA hybridize, to the resulting double strand and consequentlyno longer being available for inhibiting the biocatalytically activelabel.
 7. The method as claimed in claim 1, wherein the inhibitor is asubstance which binds reversibly to the enzyme and inhibits the enzymicactivity.
 8. The method as claimed in claim 1, wherein the DNA to beanalyzed forms, by hybridization with the immobilized DNA, a doublestrand, i.e. a double helix, on account of the complementarity of thesingle-stranded DNAs.
 9. The method as claimed in claim 1, wherein thefirst, inactive state and the second, active state of the system, andits change from_the first state to the second state, together constitutea switching function.
 10. The method as claimed in 1, wherein theswitching function of the system is effected by the DNA to be analyzedhybridizing to the immobilized DNA, as catcher.
 11. The method asclaimed in claim 9, wherein the state of the switching function of thesystem is interrogated by determining the activity of the biocatalyst.12. The method as claimed in claim 1, further-comprising: using anenzyme; forming an activatable switch from the enzyme; and reading outthe signal of the enzyme switch least one of optically andelectrochemically.
 13. The method as claimed in claim 12, wherein theenzyme switch is controlled by the catcher DNA and the DNA to beanalyzed hybridizing under stringent conditions.
 14. The method asclaimed in claim 12, wherein a product which is at least one opticallyand electrochemically detectable is synthesized, and wherein the enzymecatalyzes the conversion of an undetectable substrate into a product atleast one of optically and electrochemically detectable.
 15. The methodas claimed in claim 12, wherein the electrochemical read-out is effectedat least one of amperometrically, potentiometrically andconductometrically.
 16. The method as claimed in claim 12, wherein themeasured values of the enzyme switch are output digitally and can beread off directly.
 17. The method as claimed in claim 12, wherein theanalyte DNA concentration is effected by correlation between thequantity of enzyme product released and the quantity of DNA to beanalyzed which is hybridized.
 18. The method as claimed in claim 17,wherein the enzyme switch is deactivated by interaction of the inhibitorwith the enzyme.
 19. The method as claimed in claim 18, wherein theenzyme is inactivated if the inhibitor is bound to the enzyme and inthat the inhibitor is unavailable to the enzyme, because of the doublestrand, and the enzyme is active, when a double helix exists between thecatcher DNA and the DNA to be analyzed.
 20. An arrangement, comprising:a support on which an enzyme is immobilized at a site; a catcher DNAwhich is immobilized at the site; an inhibitor which is covalentlylinked to the catcher DNA and a substrate, with, in a first state, thecatcher DNA being folded by way of intramolecular hydrogen bonds suchthat the inhibitor inhibits the activity of the enzyme and the substrateis not transformed, and with, in a second state, the catcher DNAhybridizing with a DNA to be detected and thereby being folded such thatthe inhibitor is separated from the enzyme and the substrate istransformed, wherein the support includes integrated microelectrodes,with the enzyme being at least one of immobilized on the support, andbeing at least one of enclosed and immobilized in a polymer network inthe vicinity of the microelectrodes, and wherein at least one of theproduct and the substrate of the enzymic reaction is electrochemicallydetectable at the microelectrodes.
 21. The arrangement as claimed inclaim 20, wherein the polymer network does not interfere with theactivity of the enzyme and is permeable for the analyte DNA to beanalyzed.
 22. The arrangement as claimed in claim 20, wherein the enzymeis at least one of a phosphatase, esterase and protease.
 23. Thearrangement as claimed in claim 22, wherein the enzyme is composed of apolypeptide chain and wherein the polypeptide chain is immobilizedwithout the enzyme losing any activity.
 24. The arrangement as claimedin claim 23, wherein the enzyme is thermostable.
 25. The arrangement asclaimed in claim 24, wherein the enzyme can be produced by an expressionsystem which comprises at least one recombinant plasmid.
 26. (canceled)27. (canceled)
 28. The method as claimed in claim 1, wherein the partialdouble/single strandedness of the catcher DNA enables the inhibitor andbiocatalyst to interact.
 29. The method as claimed in claim 13, whereina product which is at least one optically and electrochemicallydetectable is synthesized, and wherein the enzyme catalyzes theconversion of an undetectable substrate into a product at least one ofoptically and electrochemically detectable.
 30. The method as claimed inclaim 13, wherein the electrochemical read-out is effected at least oneof amperometrically, potentiometrically and conductometrically.
 31. Themethod as claimed in claim 14, wherein the electrochemical read-out iseffected at least one of amperometrically, potentiometrically andconductometrically.
 32. The apparatus of claim 20, wherein at least oneof the product and the substrate of the enzymic reaction is at least oneof amperometrically, potentiometrically and conductometricallydetectable at the microelectrodes.